Methods and compositions using monoclonal antibody to human helper T cells

ABSTRACT

Hybrid cell line for production of monoclonal antibody to an antigen found on all normal human helper T cells. The hybrid is formed by fusing splenocytes from immunized CAF 1  mice with P3X63Ag8U1 myeloma cells. Diagnostic and therapeutic uses of the monoclonal antibody are also disclosed.

This is a division of our copending application Ser. No. 432,459, filedOct. 4, 1982, now U.S. Pat. No. 4,515,895, which in turn is a divisionof application Ser. No. 33,639, filed Apr. 26, 1979, now U.S. Pat. No.4,381,295.

FIELD OF THE INVENTION

This invention relates generally to new hybrid cell lines and morespecifically to hybrid cell lines for production of monoclonal antibodyto an antigen found on all normal human helper T cells, to the antibodyso produced, and to therapeutic and diagnostic methods and compositionsemploying this antibody.

DESCRIPTION OF THE PRIOR ART

The fusion of mouse myeloma cells to spleen cells from immunized mice byKohler and Milstein in 1975 [Nature 256, 495-497 (1975)] demonstratedfor the first time that it was possible to obtain a continuous cell linemaking homogeneous (so-called "monoclonal") antibody. Since this seminalwork, much effort has been directed to the production of various hybridcells (called "hybridomas") and to the use of the antibody made by thesehybridomas for various scientific investigations. See, for example,Current Topics in Microbiology and Immunology, Volume 81--"LymphocyteHybridomas", F. Melchers, M. Potter, and N. Warner, Editors,Springer-Verlag, 1978, and references contained therein; C. J.Barnstable, et al., Cell, 14, 9-20 (May, 1978); P. Parham and W. F.Bodmer, Nature 276, 397-399 (November, 1978); Handbook of ExperimentalImmunology, Third Edition, Volume 2, D. M. Wier, Editor, Blackwell,1978, Chapter 25; and Chemical and Engineering News, Jan. 1, 1979,15-17.

These references simultaneously indicate the rewards and complicationsof attempting to produce monoclonal antibody from hybridomas. While thegeneral technique is well understood conceptually, there are manydifficulties met and variations required for each specific case. Infact, there is no assurance, prior to attempting to prepare a givenhybridoma, that the desired hybridoma will be obtained, that it willproduce antibody if obtained, or that the antibody so produced will havethe desired specificity. The degree of success is influenced principallyby the type of antigen employed and the selection technique used forisolating the desired hybridoma.

The attempted production of monoclonal antibody to human lymphocytecell-surface antigens has been reported only in a few instances. See,for example, Current Topics in Microbiology and Immunology, ibid, 66-69and 164-169. The antigens used in these reported experiments werecultured human lymphoblastoid leukemia and human chronic lymphocyticleukemia cell lines. Many hybridomas obtained appeared to produceantibody to various antigens on all human cells. None of the hybridomasproduced antibody against a predefined class of human lymphocytes.

It should be understood that there are two principal classes oflymphocytes involved in the immune system of humans and animals. Thefirst of these (the thymus-derived cell or T cell) is differentiated inthe thymus from haemopoietic stem cells. While within the thymus, thedifferentiating cells are termed "thymocytes." The mature T cells emergefrom the thymus and circulate between the tissues, lymphatics, and thebloodstream. These T cells form a large proportion of the pool ofrecirculating small lymphocytes. They have immunological specificity andare directly involved in cell-mediated immune responses (such as graftrejection) as effector cells. Although T cells do not secrete humoralantibodies, they are sometimes required for the secretion of theseantibodies by the second class of lymphocytes discussed below. Sometypes of T cells play a regulating function in other aspects of theimmune system. The mechanism of this process of cell cooperation is notyet completely understood.

The second class of lymphocytes (the bone marrow-derived cells or Bcells) are those which secrete antibody. They also develop fromhaemopoietic stem cells, but their differentiation is not determined bythe thymus. In birds, they are differentiated in an organ analogous tothe thymus, called the Bursa of Fabricius. In mammals, however, noequivalent organ has been discovered, and it is thought that these Bcells differentiate within the bone marrow.

It is now recognized that T cells are divided into at least severalsubtypes, termed "helper", "suppressor", and "killer" T cells, whichhave the function of (respectively) promoting a reaction, suppressing areaction, or killing (lysing) foreign cells. These subclasses are wellunderstood for murine systems, but they have only recently beendescribed for human systems. See, for example, R. L. Evans, et al.,Journal of Experimental Medicine, Volume 145, 221-232, 1977; and L.Chess and S. F. Schlossman--"Functional Analysis of Distinct HumanT-Cell Subsets Bearing Unique Differentiation Antigens", in"Contemporary Topics in Immunobiology", O. Stutman, Editor, PlenumPress, 1977, Volume 7, 363-379.

The ability to identify or suppress classes or subclasses of T cells isimportant for diagnosis or treatment of various immunoregulatorydisorders or conditions.

For example, certain leukemias and lymphomas have differing prognosisdepending on whether they are of B cell or T cell origin. Thus,evaluation of the disease prognosis depends upon distinguishing betweenthese two classes of lymphocytes. See, for example, A. C. Aisenberg andJ. C. Long, The American Journal of Medicine, 58:300 (March, 1975); D.Belpomme, et al., in "Immunological Diagnosis of Leukemias andLymphomas", S. Thierfelder, et al., eds, Springer, Heidelberg, 1977,33-45; and D. Belpomme, et al., British Journal of Haematology, 1978,38, 85.

Certain disease states (e.g., juvenile rheumatoid arthritis, certainleukemias, and agammaglobulinemia) are associated with an imbalance of Tcell subclasses. It has been suggested that autoimmune diseasesgenerally are associated with an excess of "helper" T cells or adeficiency of certain "suppressor" T cells, while agammaglobulinemia isassociated with an excess of certain "suppressor" T cells or adeficiency of "helper" T cells. Malignancies generally are associatedwith an excess of "suppressor" T cells.

In certain leukemias, excess T cells are produced in an arrested stageof development. Diagnosis may thus depend on the ability to detect thisimbalance or excess. See, for example, J. Kersey, et al., "SurfaceMarkers Define Human Lymphoid Malignancies with Differing Prognoses" inHaematology and Blood Transfusion, Volume 20, Springer-Verlag, 1977,17-24, and references contained therein.

Acquired agammaglobulinemia, a disease state in which no immune globulinis produced, comprises at least two distinct types. In type I thefailure to produce immune globulin is due to an excess of suppressor Tcells, while in type II it is due to a lack of helper T cells. In bothtypes, there appears to be no defect or lack in the patients' B cells,the lymphocytes which are responsible for the actual secretion of theantibody; however, these B cells are being either suppressed or "nothelped", resulting in greatly decreased or absent immune globulinproduction. The type of acquired agammaglobulinemia may thus bedetermined by testing for an excess of suppressor T cells or an absenceof helper T cells.

On the therapeutic side, there is some suggestion, as yet not definitelyproven, that administration of antibodies against the subtype of T cellin excess may have therapeutic benefit in autoimmune disease ormalignancies. For example, a helper T cell cancer (certain cutaneous Tcell lymphomas and certain T cell acute lymphoblastic leukemias) may betreated by an antibody to a helper T cell antigen. Treatment ofautoimmune disease caused by an excess of helper cells may also beaccomplished in the same fashion.

Antisera against the entire class of human T cells (so-called antihumanthymocyte globulin or ATG) has been reported useful therapeutically inpatients receiving organ transplants. Since the cell-mediated immuneresponse (the mechanism whereby transplants are rejected) depends upon Tcells, administration of antibody to T cells prevents or retards thisrejection process. See, for example, Cosimi, et al., "Radomized ClinicalTrial of ATG in Cadaver Renal Allgraft Recipients: Importance of T CellMonitoring", Surgery 40:155-163 (1976) and references contained therein.

The identification and suppression of human T cell classes andsubclasses has previously been accomplished by the use of spontaneousautoantibodies or selective antisera for human T cells obtained byimmunizing animals with human T cells, bleeding the animals to obtainserum, and adsorbing the antiserum with (for example) autologous but notallogeneic B cells to remove antibodies with unwanted reactivities. Thepreparation of these antisera is extremely difficult, particularly inthe adsorption and purification steps. Even the adsorbed and purifiedantisera contain many impurities in addition to the desired antibody,for several reasons. First, the serum contains millions of antibodymolecules even before the T cell immunization. Second, the immunizationcauses production of antibodies against a variety of antigens found onall human T cells injected. There is no selective production of antibodyagainst a single antigen. Third, the titer of specific antibody obtainedby such methods is usually quite low, (e.g., inactive at dilutionsgreater than 1:100) and the ratio of specific to non-specific antibodyis less than 1/10⁶.

See, for example, the Chess and Schlossman article referred to above (atpages 365 and following) and the Chemical and Engineering News articlereferred to above, where the deficiencies of prior art antisera and theadvantages of monoclonal antibody are described.

SUMMARY OF THE INVENTION

There has now been discovered a novel hybridoma (designated OKT4) whichis capable of producing novel monoclonal antibody against an antigenfound on essentially all normal human peripheral helper T cells (about55% of normal human peripheral T cells). The antibody so produced ismonospecific for a single determinant on normal human helper T cells andcontains essentially no other anti-human immuneglobulin, in contrast toprior art antisera (which are inherently contaminated with antibodyreactive to numerous human antigens) and to prior art monoclonalantibodies (which are not monospecific for a human helper T cellantigen). Moreover, this hybridoma can be cultured to produce antibodywithout the necessity of immunizing and killing animals, followed by thetedious adsorption and purification steps necessary to obtain even theimpure antisera of the prior art.

It is accordingly one object of this invention to provide hybridomaswhich produce antibodies against an antigen found on essentially allnormal human helper T cells.

It is a further aspect of the present invention to provide methods forpreparing these hybridomas.

A further object of the invention is to provide essentially homogeneousantibody against an antigen found on essentially all normal human helperT cells.

A still further object is to provide methods for treatment or diagnosisof disease employing these antibodies.

Other objects and advantages of the invention will become apparent fromthe examination of the present disclosure.

In satisfaction of the foregoing objects and advantages, there isprovided by this invention a novel hybridoma producing novel antibody toan antigen found on essentially all normal human helper T cells, theantibody itself, and diagnostic and therapeutic methods employing theantibody. The hybridoma was prepared generally following the method ofMilstein and Kohler. Following immunization of mice with normal Erosette positive human T cells, the spleen cells of the immunized micewere fused with cells from a mouse myeloma line and the resultanthybridomas screened for those with supernatants containing antibodywhich gave selective binding to normal E rosette positive human T cells.The desired hybridomas were subsequently cloned and characterized. As aresult, a hybridoma was obtained which produces antibody (designatedOKT4) against an antigen on essentially all normal human helper T cells.Not only does this antibody react with essentially all normal humanperipheral helper T cells, but it also does not react with other normalperipheral blood lymphoid cells, including non-helper T cells. Inaddition, the cell surface antigen recognized by this antibody isdetected on approximately 80% of normal human thymocytes. Type IIacquired agammaglobulinemia patients were detected by OKT4 antibody in ablind test. The subject antibody also reacts with about 55% of Rhesusmonkey peripheral T cells.

In view of the difficulties indicated in the prior art and the lack ofsuccess reported using malignant cell lines as the antigen, it wassurprising that the present method provided the desired hybridoma. Itshould be emphasized that the unpredictable nature of hybrid cellpreparation does not allow one to extrapolate from one antigen or cellsystem to another. In fact, the present applicants have discovered thatusing a T cell malignant cell line as the antigen caused formation ofhybridomas which did not produce the desired antibody. Attempts to usepurified antigens separated from the cell surfaces were alsounsuccessful.

Both the subject hybridoma and the antibody produced thereby areidentified herein by the designation "OKT4", the particular materialreferred to being apparent from the context. The subject hybridoma OKT4was deposited in the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852, on Apr. 26, 1979, and was given the ATCCaccession number CRL 8002.

The preparation and characterization of the hybridoma and the resultantantibody will be better understood by reference to the followingdescription and Examples.

DETAILED DESCRIPTION OF THE INVENTION

The method of preparing the hybridoma generally comprises the followingsteps:

A. Immunizing mice with E rosette positive purified normal humanperipheral T cells. While it has been found that female CAF₁ mice arepreferred, it is contemplated that other mouse strains could be used.The immunization schedule and T cell concentration should be such as toproduce useful quantities of suitably primed splenocytes. Threeimmunizations at fourteen day intervals with 2×10⁷ cells/mouse/injectionin 0.2 ml phosphate buffered saline has been found to be effective.

B. Removing the spleens from the immunized mice and making a spleensuspension in an appropriate medium. About one ml of medium per spleenis sufficient. These experimental techniques are well-known.

C. Fusing the suspended spleen cells with mouse myeloma cells from asuitable cell line by the use of a suitable fusion promoter. Thepreferred ratio is about 5 spleen cells per myeloma cell. A total volumeof about 0.5-1.0 ml of fusion medium is appropriate for about 10⁸splenocytes. Many mouse myeloma cell lines are known and available,generally from members of the academic community or various depositbanks, such as the Salk Institute Cell Distribution Center, La Jolla,Calif. The cell line used should preferably be of the so-called "drugresistant" type, so that unfused myeloma cells will not survive in aselective medium, while hybrids will survive. The most common class is8-azaguanine resistant cell lines, which lack the enzyme hypoxanthineguanine phophoribosyl transferase and hence will not be supported by HAT(hypoxanthine, aminopterin, and thymidine) medium. It is also generallypreferred that the myeloma cell line used be of the so-called"non-secreting" type, in that it does not itself produce any antibody,although secreting types may be used. In certain cases, however,secreting myeloma lines may be preferred. While the preferred fusionpromoter is polyethylene glycol having an average molecular weight fromabout 1000 to about 4000 (commercially available as PEG 1000, etc.),other fusion promoters known in the art may be employed.

D. Diluting and culturing in separate containers, the mixture of unfusedspleen cells, unfused myeloma cells, and fused cells in a selectivemedium which will not support the unfused myeloma cells for a timesufficient to allow death of the unfused cells (about one week). Thedilution may be a type of limiting one, in which the volume of diluentis statistically calculated to isolate a certain number of cells (e.g.,1-4) in each separate container (e.g., each well of a microtiter plate).The medium is one (e.g., HAT medium) which will not support thedrug-resistant (e.g., 8-azaguanine resistant) unfused myeloma cell line.Hence, these myeloma cells perish. Since the unfused spleen cells arenon-malignant, they have only a finite number of generations. Thus,after a certain period of time (about one week) these unfused spleencells fail to reproduce. The fused cells, on the other hand, continue toreproduce because they possess the malignant quality of the myelomaparent and the ability to survive in the selective medium of the spleencell parent.

E. Evaluating the supernatant in each container (well) containing ahybridoma for the presence of antibody to E rosette positive purifiedhuman T cells.

F. Selecting (e.g., by limiting dilution) and cloning hybridomasproducing the desired antibody.

Once the desired hybridoma has been selected and cloned, the resultantantibody may be produced in one of two ways. The purest monoclonalantibody is produced by in vitro culturing of the desired hybridoma in asuitable medium for a suitable length of time, followed by recovery ofthe desired antibody from the supernatant. The suitable medium andsuitable length of culturing time are known or are readily determined.This in vitro technique produces essentially monospecific monoclonalantibody, essentially free from other specific antihuman immuneglobulin. There is a small amount of other immune globulin present sincethe medium contains xenogeneic serum (e.g., fetal calf serum). However,this in vitro method may not produce a sufficient quantity orconcentration of antibody for some purposes, since the concentration ofmonoclonal antibody is only about 50 μg/ml.

To produce a much greater concentration of slightly less pure monoclonalantibody, the desired hybridoma may be injected into mice, preferablysyngenic or semisyngenic mice. The hybridoma will cause formation ofantibody-producing tumors after a suitable incubation time, which willresult in a high concentration of the desired antibody (about 5-20mg/ml) in the bloodstream and peritoneal exudate (ascites) of the hostmouse. Although these host mice also have normal antibodies in theirblood and ascites, the concentration of these normal antibodies is onlyabout 5% of the monoclonal antibody concentration. Moreover, since thesenormal antibodies are not antihuman in their specificity, the monoclonalantibody obtained from the harvested ascites or from the serum isessentially free of any contaminating antihuman immune globulin. Thismonoclonal antibody is high titer (active at dilutions of 1:50,000 orhigher) and high ratio of specific to non-specific immune globulin(about 1/20). Immune globulin produced incorporating the κ light myelomachains are non-specific, "nonsense" peptides which merely dilute themonoclonal antibody without detracting from its specificity.

EXAMPLE I Production of Monoclonal Antibodies

A. Immunization and Somatic Cell Hybridization

Female CAF₁ mice (Jackson Laboratories; 6-8 weeks old) were immunizedintraperitoneally with 2×10⁷ E rosette purified T cells in 0.2 ml ofphosphate buffered saline at 14-day intervals. Four days after the thirdimmunization, spleens were removed from the mice, and a single cellsuspension was made by pressing the tissue through a stainless steelmesh.

Cell fusion was carried out according to the procedure developed byKohler and Milstein. 1×10⁸ splenocytes were fused in 0.5 ml of a fusionmedium comprising 35% polyethylene glycol (PEG 1000) and 5%dimethylsulfoxide in RPMI 1640 medium (Gibco, Grand Island, N.Y.) with2×10⁷ P3X63Ag8Ul myeloma cells supplied by Dr. M. Scharff, AlbertEinstein College of Medicine, Bronx, N.Y. These myeloma cells secreteIgG₁ κ light chains.

B. Selection and Growth of Hybridoma

After cell fusion, cells were cultured in HAT medium (hypoxanthine,aminopterin, and thymidine) at 37° C. with 5% CO₂ in a humid atmosphere.Several weeks later, 40 to 100 μl of supernatant from culturescontaining hybridomas were added to a pellet of 10⁶ peripherallymphocytes separated into E rosette positive (E⁺) E rosette negative(E⁻) populations, which were prepared from blood of healthy human donorsas described by Mendes (J. Immunol. 111:860, 1973). Detection of mousehybridoma antibodies binding to these cells was determined byradioimmunoassay and/or indirect immunofluorescence. In the firstmethod, the cells were initially reacted with 100 μl ofaffinity-purified ¹²⁵ I goat-anti-mouse IgG (10⁶ cpm/μg; 500 μg/μl).(Details of iodination of goat-anti-mouse IgG were described by Kung, etal., J. Biol. Chem. 251(8):2399, 1976). Alternatively, cells incubatedwith culture supernatants were stained with a fluorescinatedgoat-anti-mouse IgG (G/M FITC) (Meloy Laboratories, Springfield, Va.;F/p=2.5) and the fluorescent antibody-coated cells were subsequentlyanalyzed on the Cytofluorograf FC200/4800A (Ortho Instruments, Westwood,Mass.) as described in Example III. Hybridoma cultures containingantibodies reacting specifically with E⁺ lymphocytes (T cells) wereselected and cloned. Subsequently, the clones were transferredintraperitoneally by injecting 1×10.sup. 7 cells of a given clone (0.2ml volume) into CAF₁ mice primed with 2,6,10,14-tetramethylpentadecane,sold by Aldrich Chemical Company under the name Pristine. The malignantascites from these mice were then used to characterize lymphocytes asdescribed below in Example II. The subject hybrid antibody OKT4 wasdemonstrated by standard techniques to be of IgG₂ sublass and to fixcomplement.

EXAMPLE II Characterization of OKT4 Reactivity

A. Isolation of Lymphocyte Populations

Human peripheral blood mononuclear cells were isolated from healthyvolunteer donors (ages 15-40) by Ficoll-Hypaque density gradientcentrifugation (Pharmacia Fine Chemicals, Piscataway, N.J.) followingthe technique of Boyum, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97): 77,1968. Unfractionated mononuclear cells were separated into surface Ig⁺(B) and Ig⁻ (T plus Null) populations by Sephadex G-200 anti-F(ab')₂column chromatography as previously described by Chess, et al., J.Immunol. 113:1113 (1974). T cells were recovered by E rosetting the Ig⁻population with 5% sheep erythrocytes (Microbiological Associates,Bethesda, Md.). The rosetted mixture was layered over Ficoll-Hypaque andthe recovered E⁺ pellet treated with 0.155M NH₄ Cl (10 ml per 10⁸cells). The T cell population so obtained was <2% EAC rosette positiveand >95% E rosette positive as determined by standard methods. Inaddition, the non-rosetting Ig⁻ (Null cell) population was harvestedfrom the Ficoll interface. This latter population was <5% E⁺ and ≦2%sIg⁺. The surface Ig⁺ (B) population was obtained from the SephadexG-200 column following elution with normal human gamma globulin aspreviously described. This population was >95% surface Ig⁺ and <5% E⁺.

Normal human macrophages were obtained from the mononuclear populationby adherence to polystyrene. Thus, mononuclear cells were resuspended infinal culture media (RPMI 1640, 2.5 mM HEPES[4-(2-hydroxyethyl)-1-piperazinepropane sulfonic acid] buffer, 0.5%sodium bicarbonate, 200 mM L-glutamine, and 1% penicillinstreptomycin,supplemented with 20% heat-inactivated human AB serum) at aconcentration of 2×10⁶ cells and incubated in plastic petri dishes(100×20 mm) (Falcon Tissue Culture Dish; Falcon, Oxnard, Calif.) at 37°C. overnight. After extensive washing to remove non-adherent cells, theadherent population was detached by brisk washing with cold serum-freemedium containing 2.5 mM EDTA and occasional scraping with the rubbertip of a disposable syringe plunger. Greater than 85% of the cellpopulation was capable of ingesting latex particles and had morphologiccharacteristics of monocytes by Wright-Giemsa staining.

B. Normal Thymus

Normal human thymus gland was obtained from patients aged two months to14 years undergoing corrective cardiac surgery. Freshly obtainedportions of the thymus gland were immediately placed in 5% fetal calfserum in medium 199 (Gibco), finely minced with forceps and scissors,and subsequently made into single cell suspensions by being pressedthrough wire mesh. The cells were next layered over Ficoll-Hypaque andspun and washed as previously described in section A above. Thethymocytes so obtained were >95% viable and ≧90% E rosette positive.

C. Cell Lines

Epstein-Barr Virus (EBV) transformed B cell lines from four normalindividuals (Laz 007, Laz 156, Laz 256, and SB) and T cell lines CEM,HJD-1, Laz 191, and HM1 established from leukemic patients were providedby Dr. H. Lazarus, Sidney Farber Cancer Institute, Boston, Mass.

D. T Acute Lymphoblastic Leukemia (T-ALL) Cells and T Chronic LymphaticLeukemia (T-CLL) Cells

Leukemia cells were obtained from 12 patients with T-ALL. Theseindividuals' cells had previously been determined to be of T celllineage by their spontaneous rosette formation with sheep erythrocytes(>20% E⁺) and reactivity with T cell specific hetero-antisera, anti-HTL(anti-B.K.) and A99, as previously described by Schlossman, et al.,Proc. Nat. Acad. Sci. 73:1288 (1976). Tumor cells from three individualswere reactive (TH₂ ⁺) with rabbit and/or equine anti-TH₂ while cellsfrom the remaining nine were non-reactive (TH₂ ⁻). Leukemic cells fromtwo patients with TH₂ ⁻ T-CLL were also utilized. Both acute and chronicT cell leukemia cells were cryopreserved in -196° C. vapor phase liquidnitrogen in 10% dimethylsulfoxide and 20% AB human serum until the timeof surface characterization. The tumor populations analyzed were >90%blasts by Wright-Giemsa morphology in all instances.

EXAMPLE III Cytofluorographic Analysis and Cell Separation

Cytofluorographic analysis of all cell populations was performed byindirect immunofluorescence with fluorescein-conjugated goat-anti-mouseIgG (G/M FITC) (Meloy Laboratories) on a Cytofluorograf FC200/4800A(Ortho Instruments). In brief, 1-2×10⁶ cells were treated with 0.15 mlOKT4 at a 1:1000 dilution, incubated at 4° C. for 30 minutes, and washedtwice. The cells were then reacted with 0.15 ml of a 1:40 dilution G/MFITC at 4° C. for 30 minutes, centrifuged, and washed three times. Thesecells were then analyzed on the Cytofluorograf and the intensity offluorescence per cell recorded on a pulse height analyzer. A similarpattern of reactivity was observed at a dilution of 1:50,000, butfurther dilution caused loss of reactivity. Background staining wasobtained by substituting a 0.15 ml aliquot of 1:1000 ascites from aBalb/cJ mouse intraperitoneally immunized with a non-producing hybridclone.

In experiments designed to separate OKT4⁺ and OKT4⁻ cells, 100×10⁶unfractionated mononuclear cells or thymocytes were labeled with 4 ml ofa 1:1000 dilution of OKT4 and developed with G/M FITC. An identicalstaining approach was utilized to prepare human T cells isolated as inExample IIA above. Utilizing a fluorescence activated cell sorter(FACS-I) (Becton-Dickinson, Mountain View, Calif.), lymphocytes wereseparated into OKT4⁺ and OKT4⁻ populations. Post sort viability was >95%by Trypan blue exclusion in all instances. Purity of all separatedpopulations was ≧95%.

EXAMPLE IV Analysis of FACS Separated OKT4⁺ and OKT4⁺ Subsets withEquine Anti-TH₂

OKT4⁺ and OKT4⁻ T cells were separated on FACS and placed in culture at2×10⁶ cells per ml in RPMI 1640 (Grand Island Biological Company),containing 20% human AB serum, 1% penicillin-streptomycin, 200 mML-glutamine, 25 mM HEPES buffer (Microbiological Associates), and 0.5%sodium bicarbonate. After 24 hours in a 5% CO₂ humid atmosphere at 37°C., 1-2×10⁶ cells of each population were reacted with equine anti-TH₂and stained with fluorescein-conjugated IgG fraction rabbit anti-horseIg (Cappel Laboratories, Downington, Pa.) as described by Reinherz andSchlossman, J. Immunol. 122:1335-1341 (1979). Background staining wasdetermined by substituting normal horse IgG for specific antibody andstaining as above.

EXAMPLE V Functional Studies

A. Proliferative Studies

The mitogenic response of the unseparated and FACS-fractionated lymphoidcells was tested in microculture to optimal and suboptimal doses ofConcanavalin A (Con A) (Calbiochem, La Jolla, Calif.) andPhytohemagglutinin (PHA) (Burroughs-Wellcome Company, Greenville, N.C.).Alloantigen proliferative response was measured concurrently for thesesame populations using mitomycin c treated Laz 156, an EBV transformedhuman B lymphoblastoid cell line, as a stimulus. Proliferation totetanus toxoid (Massachusetts Department of Public Health BiologicalLaboratories, Boston, Mass.) was tested utilizing 10 μg/ml finalconcentration. Herpes-Zoster antigen was kindly provided by Dr. JohnZaia (Harvard Medical School, Boston, Mass.) and utilized at a 1:6dilution. Five percent macrophages obtained in the manner describedabove were added to all populations at the initiation of in vitrocultures. Mitogen stimulated cultures were pulsed after four days with0.2 μCi of ³ H-thymidine (³ H-TdR) (1.9 Ci/mM specific activity)(Schwartz-Mann, Division of Becton, Dickinson, Orangeburg, N.Y.) andharvested 18 hours later on a MASH II apparatus (MicrobiologicalAssociates). ³ H-TdR incorporation was measured in a PackardScintillation Counter (Packard Instrument Company, Downer's Grove,Ill.). Background ³ H-TdR incorporation was obtained by substitutingmedia for mitogen. Soluble antigen and alloantigen stimulated cultureswere pulsed after five days with ³ H-TdR for 18 hours, harvested, andcounted as above.

B. Cytotoxicity Studies

Sensitization cultures for cell-mediated lympholysis (CML) wereestablished by placing unfractionated T cells, FACS-separated OKT4⁺ andOKT4⁻ T cell subsets, or different ratios of recombined OKT4⁺ and OKT4⁻T cells with mitomycin-treated stimulator cells all at 2×10⁶ cells perml in multiple microtitre plate wells. At the end of five days,non-viable cells were removed by Ficoll-Hypaque centrifugation. Theseunfractionated and fractionated T cell populations were then added to ⁵¹Cr sodium chromate labeled target cells and specific chromium releasedetermined after a six-hour cell incubation. In other experiments,unfractionated T cells were sensitized with mitomycin-treated stimulatorcells as above and then fractionated into OKT4⁺ and OKT4⁻ T cell subsetson FACS after five days in MLC and specific chromium release determined.Percent cytotoxicity was determined with the following formula: ##EQU1##

All samples were performed in triplicate and results expressed as themean. Spontaneous release was less than 20% of maximal lysis in allcases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the fluorescence pattern obtained on the Cytofluorografafter reacting normal human peripheral T cells with OKT4 at a 1:1000dilution and G/M FITC. For comparison, results with monoclonalantibodies OKT1 and OKT3 are shown under equivalent conditions in FIGS.1-5.

FIG. 2 shows the fluorescence pattern obtained on the Cytofluorografafter reacting human thymocytes with OKT4 and G/M FITC.

FIG. 3 shows the fluorescence pattern obtained on the Cytofluorografafter reacting leukemic cells from B cell chronic lymphoblastic leukemiapatients with OKT4 and G/M FITC.

FIG. 4 shows the fluorescence pattern obtained on the Cytofluorografafter reacting the human T cell line HJD-1 with OKT4 and G/M FITC.

FIG. 5 shows the fluorescence pattern obtained on the Cytofluorografafter reacting the human T cell line CEM with OKT4 and G/M FITC.

FIG. 6 shows the results of reaction of T cell populations with equineanti-TH₂ serum.

FIG. 7 shows the cytotoxic capacity of unfractionated T cells and T cellsubsets. Percent specific lysis is shown on the ordinate andeffector/target (E/T) ratio is shown on the abscissa.

The production of the hybridoma and the production and characterizationof the resulting monoclonal antibody were conducted as described in theabove Examples. Although large quantities of the subject antibody wereprepared by injecting the subject hybridoma intraperitoneally into miceand harvesting the malignant ascites, it is clearly contemplated thatthe hybridoma could be cultured in vitro by techniques well-known in theart and the antibody removed from the supernatant.

As shown in FIG. 1, approximately 45% of the human peripheral blood Tcell population of a given normal individual is reactive with OKT4,whereas the entire B cell, null cell, and macrophage populationsisolated from the same individual are unreactive with OKT4. Similarresults were obtained on populations of lymphocytes from fifteen othernormal individuals. The monoclonal antibody is thus characterized inthat it is reactive with an antigen contained on the surface ofapproximately 55% of normal human peripheral T cells, while beingunreactive with any antigens on the surface of the other three celltypes discussed above. As will be discussed below, the OKT4⁺ portion ofthe human peripheral T cell population is the subclass of helper Tcells. This differential reactivity is one test by which the subjectantibody OKT4 may be detected and distinguished from other antibodies.

As shown in FIG. 2, approximately 80% of normal human thymocytes from asix-month old infant are reactive with OKT4. Similar results (about 80%reactivity) were obtained using six additional thymus specimens fromnormal individuals two months to 19 years of age. The pattern ofreactivity in FIG. 2 provides a second method of detecting the subjectantibody OKT4 and distinguishing it from other antibodies.

As shown in FIG. 3, the subject antibody is unreactive with leukemiccells from B cell chronic lymphoblastic leukemia.

A further characterization of the subject antibody OKT4 is shown by thereactivity to various human T cell lines illustrated in FIGS. 4 and 5.As can be seen, the reactivity of the subject antigen to human T celllines was heterogeneous, being strong for the line CEM, and nonexistentfor the line HDJ-1. There was also no reactivity with the cell lines Laz191 and HM1. This differential reactivity of OKT4 to variousreadily-available human T cell lines provides yet another method ofcharacterizing and describing the subject antibody.

FIG. 6 illustrates the reactivity of OKT4 separated subsets withanti-TH₂. About 25% of the unfractionated T cell population is reactivewith anti-TH₂. In contrast, the OKT4⁺ population contains no cells whichare reactive with anti-TH₂, while the OKT4⁻ population is predominantlyTH₂ ⁺ and contains all the TH₂ ⁺ cells found in the unfractionated Tcell population. This indicated that the TH₂ ⁺ and the OKT4⁺ subsets arereciprocal and distinct from one another.

FIG. 7 illustrates the cytotoxic capacity of unfractionated, OKT4⁺, andOKT4⁻ T cells. The OKT4⁺ population is only minimally cytotoxic, whilethe degree of killing mediated by the OKT4⁻ population is greater thanfor the unfractionated T cell population. The differential reactivitiesof OKT4⁺ and OKT4⁻ T cell populations shown in FIGS. 6 and 7 provide afurther means of characterizing the subject antibody.

The results illustrated in FIGS. 1-5 are summarized and augmented withadditional data in Table I below. The Table compares monoclonalantibodies produced by hybridomas designated OKT1, OKT3, and OKT4 (whichlast is the subject of the present application). In addition to the datain FIGS. 1-5, Table I also demonstrates that OKT4 is unreactive withnormal human peripheral B cells, null cells and macrophages, as well asleukemic cells from T cell and null cell acute lymphoblastic leukemiapatients and EBV transformed B cell lines. In contrast to OKT1, OKT4does not react with leukemic cells from T cell chronic lymphoblasticleukemia patients.

Functional studies were conducted on lymphoid populations which had beenseparated on a fluorescence activated cell separator (FACS). The resultsof these studies are shown in Tables II through IV below and providefurther support for the previously-described characterization of thesubject monoclonal antibody.

In these studies, an unfractionated T cell population was treated with a1:1000 dilution of OKT4 and G/M FITC and separated on the FACS in OKT4⁺and OKT4⁻ subsets. Given the purity of the populations obtained (greaterthan or equal to 95%), 5% macrophages were added to the separatedpopulations prior to in vitro culture. The unfractionated T cellpopulation and isolated OKT4⁺ and OKT4⁻ T cell subsets were thenstimulated with PHA, Con A, soluble antigens, and alloantigens to assesstheir in vitro proliferative responses.

The proliferative response of the unfractionated T cell populations toPHA and Con A is shown in Table II. A maximal proliferative response bythe unfractionated T cell population is obtained with 1 μg of PHA per10⁶ cells with diminished responses occurring at 0.5 μg and 0.1 μg ofPHA per 10⁶ cells. Treatment of the unfractionated T cells with OKT4 andgoat-mouse FITC without subsequent fractionation did not alter theproliferative response. In contrast, differences in response to PHA wereobtained with the separated OKT4⁺ and OKT4⁻ T cell subset. The OKT4⁺population of cells responded to all doses of PHA in a fashion similarto the unseparated T cell population. However, the proliferativeresponse of OKT4⁻ cells was significantly less at all doses of PHAtested. Furthermore, at a dose of PHA of 0.1 μg per 10⁶ cells, the OKT4⁻T cells did not proliferate at all, whereas the OKT4⁺ T cell subset andunfractionated cells were still responsive. The proliferative responseof these subsets to Con A, on the other hand, was similar and the twosubsets of cells could not be distinguished from one another or theunfractionated T cell population.

The responses to alloantigen in MLC and to soluble antigens were nextexamined. As shown in Table III, the unfractionated T cell population,the unfractionated T cell population treated with OKT4 and G/M FITC, andboth the OKT4⁺ and OKT4⁻ T cell subsets responded in a similar fashionin MLC against Laz 156. It should be noted, however, that the OKT4⁻cells from two of six individuals tested, although proliferatingsignificantly in MLC, incorporated less ³ H-TdR than their respectiveOKT4⁺ subset (data not shown). In contrast, proliferative responses tosoluble antigens provided the clearest distinction between the subsets.In all cases tested, the OKT4⁺ T cell subset proliferated well to thesoluble antigens, tetanus toxoid and Herpes-Zoster, whereas the OKT4⁻ Tcell subset was virtually unresponsive.

Table IV shows that the OKT4⁻ subset of T cells cannot generate much, ifany, cytolysis when it is sensitized alone in MLC. Thus, although theOKT4⁻ T cells became cytotoxic/effectors in the unfractionatedallosensitized T cell population, by itself it could not be induced tomediate CML despite its response in MLC. Moreoever, when the OKT4⁺population was sensitized in MLC in the absence of the OKT4⁻ T cells,they could mediate a moderate, but significant, lysis in MLC. However,the recombined mixture of OKT4⁺ and OKT4⁻ T cells effected a maximalcytolysis, not unlike that of the unseparated T cell population. Thesefindings demonstrate that the OKT4⁻ subset cannot effect a maximalcytotoxic response alone, but requires help from the OKT4⁺ population.This T-T interaction is analogous to the T cell help provided by the TH₂⁻ T cell subset to the TH₂ ⁺ T cell subset in generating maximalcytotoxicity.

According to the present invention there are provided a hybridomacapable of producing antibody against an antigen found on essentiallyall normal human helper T cells, a method for producing this hybridoma,monoclonal antibody against an antigen found on essentially all humanhelper T cells, methods for producing the antibody, and methods andcompositions for treatment or diagnosis of disease employing thisantibody.

Although only a single hybridoma producing a single monoclonal antibodyagainst human helper T cell antigen is described, it is contemplatedthat the present invention encompasses all monoclonal antibodiesexhibiting the characteristics described herein. It was determined thatthe subject antibody OKT4 belongs to the subclass IgG₂, which is one offour subclasses of murine IgG. These subclasses of immune globulin Gdiffer from one another in the so-called "fixed" regions, although anantibody to a specific antigen will have a so-called "variable" regionwhich is functionally identical regardless of which subclass of immuneglobulin G it belongs to. That is, a monoclonal antibody exhibiting thecharacteristic described herein may be of subclass IgG₁, IgG₂ a, IgG₂ b,or IgG₃, or of classes IgM, IgA, or other known Ig classes. Thedifferences among these classes or subclasses will not affect theselectivity of the reaction pattern of the antibody, but may affect thefurther reaction of the antibody with other materials, such as (forexample) complement or anti-mouse antibodies. Although the subjectantibody is specifically IgG₂, it is contemplated that antibodies havingthe patterns of reactivity illustrated herein are included within thesubject invention regardless of the immune globulin class or subclass towhich they belong.

Further included within the subject invention are methods for preparingthe monoclonal antibodies described above employing the hybridomatechnique illustrated herein. Although only one example of a hybridomais given herein, it is contemplated that one skilled in the art couldfollow the immunization, fusion, and selection methods provided hereinand obtain other hybridomas capable of producing antibodies having thereactivity characteristics described herein. Since the individualhybridoma produced from a known mouse myeloma cell line and spleen cellsfrom a known species of mouse cannot be further identified except byreference to the antibody produced by the hybridoma, it is contemplatedthat all hybridomas producing antibody having the reactivitycharacteristics described above are included within the subjectinvention, as are methods for making this antibody employing thehybridoma.

Further aspects of the invention are methods of treatment or diagnosisof disease employing the monoclonal antibody OKT4 or any othermonoclonal antibody exhibiting the pattern of reactivity providedherein. The subject antibody may be used to detect type II acquiredagammaglobulinemia by reacting a T cell composition from an individualwith OKT4 antibody. Helper T cell absence or deficiency will beindicated by the presence of less than 55% of the total peripheral Tcell population which reacts with OKT4. This test may also be used todetect helper T cell defects or excesses generally. Treatment of helperT cell cancers may be accomplished by administration of atherapeutically effective amount of OKT4 antibody to an individual inneed of such treatment. By selective reaction with helper T cellantigen, the effective amount of OKT4 antibody will reduce the excess ofhelper T cells, thus ameliorating the effects of the helper T cellmalignancy. Autoimmune disease caused by an excess of helper T cells mayalso be treated by administration of a therapeutically effective amountof OKT4 antibody to an individual in need of such treatment. Diagnosticand therapeutic compositions comprising effective amounts of OKT4antibody in admixture with diagnostically or pharmaceutically acceptablecarriers, respectively, are also included within the present invention.

                  TABLE I                                                         ______________________________________                                        MONOCLONAL ANTIBODY                                                           REACTIVITY AND PROPERTIES                                                                    Monoclonal Antibodies                                                         OKT1    OKT3      OKT4                                         ______________________________________                                        % Reactivity With:                                                            Peripheral T-cells (10 samples)                                                                >95%      >95%       55%                                     Peripheral B-cells (10 samples)                                                                 <2%       <2%      <2%                                      Peripheral Null cells                                                                           <2%       <2%      <2%                                      (10 samples)                                                                  Thymocytes* (8 samples)                                                                        5-10%     5-10%      80%                                     Reactivity With:                                                              T-chronic lymphatic Leukemia                                                                   +         +(1);-(2) -                                        (3 cases)                                                                     T-acute lymphatic Leukemia                                                                     -         -         -                                        (8 cases)                                                                     Null acute lymphatic Leukemia                                                                  -         -         -                                        (15 cases)                                                                    B-chronic lymphatic Leukemia                                                                   +(4);-(2) -         -                                        (6 cases)                                                                     B-cell lines.sup.+ (4)                                                                         -         -         -                                        T-cell lines.sup.+                                                            HJD-1            +         (±)    -                                        CEM              +         -         +                                        Laz 191          +         -         -                                        HM1              +         -         -                                        IgG Subclass     IgG.sub.1 IgG.sub.2 IgG.sub.2                                Complement fixation                                                                            -         +         +                                        ______________________________________                                         *From patients aged 2 months to 18 years                                      .sup.+ Obtained from Dr. H. Lazarus, Sidney Farber Cancer Center. B cell      lines Laz 256, 156, 007 and SB obtained by EpsteinBarr virus                  transformation of human peripheral B cells and HJD1, CEM, Laz 191, and HM     established from leukemic patients.                                      

                                      TABLE II                                    __________________________________________________________________________    MITOGEN RESPONSIVENESS OF UNFRACTIONATED AND                                  MONOCLONAL ANTIBODY SEPARATED T CELL SUBSETS                                  Proliferative    T Cells Treated with                                         Stimulus                                                                              T Cells  OKT4 and G/M FITC                                                                         OKT4.sup.+  T Cells                                                                    OKT4.sup.-  T Cells                     __________________________________________________________________________    PHA (1 μg/10.sup.6                                                                 98,876                                                                            ± 3,061                                                                         99,780                                                                              ± 5,615                                                                          109,643                                                                           ± 11,043                                                                        23,841                                                                            ± 890                            cells)                                                                        PHA (0.5 μg/                                                                       18,082                                                                            ± 3,588                                                                         17,423                                                                              ± 2,623                                                                          15,202                                                                            ± 603                                                                           3,185                                                                             ± 492                            10.sup.6 cells)                                                               PHA (0.1 μg/                                                                       4,215                                                                             ± 386                                                                           4,317 ± 523                                                                            4,999                                                                             ± 677                                                                           72  ± 11                             10.sup.6 cells)                                                               Con A (250 μg/                                                                     199,310                                                                           ± 14,317                                                                        192,158                                                                             ± 14,258                                                                         159,560                                                                           ± 2,619                                                                         174,992                                                                           ± 20,179                         10.sup.6 cells)                                                               Con A (125 μg/                                                                     100,726                                                                           ± 8,864                                                                         92,633                                                                              ± 6,780                                                                          97,003                                                                            ± 9,089                                                                         82,199                                                                            ± 6,154                          10.sup.6 cells)                                                               Con A (50 μg/                                                                      50,673                                                                            ± 6,155                                                                         52,317                                                                              ± 7,813                                                                          49,329                                                                            ± 4,110                                                                         38,842                                                                            ± 6,735                          10.sup.6 cells)                                                               Media Control                                                                         63  ± 2                                                                             57    ± 6                                                                              167 ± 8                                                                             69  ± 12                             __________________________________________________________________________

                                      TABLE III                                   __________________________________________________________________________    PROLIFERATIVE RESPONSE OF UNFRACTIONATED T CELLS AND                          MONOCLONAL ANTIBODY SEPARATED T CELL SUBSETS                                  TO SOLUBLE ANTIGENS AND ALLOANTIGENS                                          Proliferative    T Cells Treated with                                         Stimulus                                                                              T Cells  OKT4 and G/M FITC                                                                         OKT4.sup.+  T Cells                                                                    OKT4.sup.-  T Cells                     __________________________________________________________________________    Experiment #1                                                                 Laz 156.sub.m                                                                         150,304                                                                           ± 6,966                                                                         149,810                                                                             ± 7,630                                                                          173,061                                                                           ± 4,336                                                                         167,087                                                                           ± 5,228                          Tetanus Toxoid                                                                        18,046                                                                            ± 271                                                                           19,947                                                                              ± 3,214                                                                          19,660                                                                            ± 2,348                                                                         516 ± 69                             Herpes-Zoster                                                                         47,413                                                                            ± 3,208                                                                         40,774                                                                              ± 4,305                                                                          55,785                                                                            ± 10,608                                                                        188 ± 129                            Media   166 ± 22                                                                            180   ± 35                                                                             220 ± 10                                                                            106 ± 13                             Experiment #2                                                                 Laz 156.sub.m                                                                         67,265                                                                            ± 3,466                                                                         68,399                                                                              ± 6,154                                                                          97,615                                                                            ± 4,361                                                                         67,483                                                                            ± 4,792                          Tetanus Toxoid                                                                        16,853                                                                            ± 1,033                                                                         18,597                                                                              ± 1,643                                                                          17,222                                                                            ± 911                                                                           206 ± 23                             Herpes-Zoster                                                                         23,629                                                                            ± 3,677                                                                         22,889                                                                              ±  1,956                                                                         25,951                                                                            ± 2,609                                                                         169 ± 25                             Media   167 ± 43                                                                            289   ± 57                                                                             298 ± 41                                                                            31  ± 8                              __________________________________________________________________________

                  TABLE IV                                                        ______________________________________                                        EVIDENCE FOR T-- T INTERACTIONS IN THE                                        GENERATION OF MAXIMAL CYTOTOXICITY IN CML                                                    % Specific Lysis.sup.+                                                        Experiment #1                                                                           Experiment #2                                        Responder Population                                                                           E/T Ratio:                                                   Sensitized to Laz 156.sub.m                                                                    20:1    5:1       20:1                                                                              5:1                                    ______________________________________                                        Unfractionated T Cells                                                                          40.sup.+                                                                             --        23  11.9                                   Unfractionated T Cells                                                                         44      24        25  13                                     Treated with OKT4 +                                                           G/M FITC                                                                      OKT4.sup.-  T Cells                                                                             6       3        10.9                                                                              6.7                                    OKT4.sup.+  T Cells                                                                            26      14        18.3                                                                              12.3                                   OKT4.sup.+  and OKT4.sup.-  T                                                                  48      31        31  16                                     Cells.sup.++                                                                  ______________________________________                                         .sup.+ S.R. less than 20% in all cases and S.D. less than or equal to 10%     at all E/T ratios. Lysis by unsensitized T cells was less than or equal t     3% for all populations tested.                                                .sup.++ OKT4.sup.+  and OKT4.sup.-  recombined at 1:1 ratios.            

What is claimed is:
 1. A therapeutic composition of matter comprising,in admixture with a pharmaceutically-acceptable carrier, an amount ofmouse monoclonal antibody effective to reduce the amount of helper Tcells in an individual having an excess of said helper T cells, saidantibody reacting with essentially all normal human peripheral helper Tcells but not with normal human peripheral B cells, null cells,macrophages.
 2. A therapeutic composition of matter comprising, inadmixture with a pharmaceutically-acceptable carrier, an amount of mousemonoclonal antibody effective to reduce the amount of helper T cells inan individual having an excess of said helper T cells, said antibodyreacting with essentially all normal human peripheral helper T cells. 3.A therapeutic composition of matter comprising, in admixture with apharmaceutically-acceptable carrier, an amount of monoclonal antibodyeffective to reduce the amount of helper T cells in an individual havingan excess of said helper T cells, said monoclonal antibody beingproduced from a hybridoma formed by fusion of cells from a mouse myelomaline and spleen cells from a mouse previously immunized with human Tcells, said antibody reacting with essentially all normal humanperipheral helper T cells but not with normal human peripheral B cells,null cells, or macrophages.
 4. A therapeutic composition of mattercomprising, in admixture with a pharmaceutically-acceptable carrier, anamount of a monoclonal antibody effective to reduce the amount of helperT cells in an individual having an excess of said helper T cells, saidmonoclonal antibody being produced by a hybridoma formed by fusion ofcells from a mouse myeloma line and spleen cells from a mouse previouslyimmunized with human T cells, said monoclonal antibody reacting withessentially all normal human peripheral helper T cells.
 5. A therapeuticcomposition of matter comprising, in admixture with a pharmaceuticallyacceptable carrier, an amount of monoclonal antibody effective to reducethe amout of helper T cells in an individual having an excess of saidhelper T cells, which antibody:(a) reacts with essentially all normalhuman peripheral helper T cells (being about 55% of all normal humanperipheral T cells), but not with normal human peripheral B cells, nullcells or macrophages; (b) reacts with about 80% of normal humanthymocytes; (c) does not react with leukemic cells from humans with Tcell chronic lymphoblastic leukemia, B cell chronic lymphoblasticleukemia, T cell acute lymphoblastic leukemia, or null cell acutelymphoblastic leukemia; (d) reacts with the human T cell line CEM, butnot with HJD-1, Laz 191, or HM1; (d) does not react with Epstein-Barrvirus-transformed human B cell lines Laz 007, Laz 156, Laz 256, or SB;(f) reacts with about 55% of Rhesus monkey peripheral T cells; (g) fixescomplement; and (h) defines a T cell population which is unreactive withanti-TH2 serum, and is only minimally cytotoxic.
 6. A diagnosticcomposition for detection of human helper T cells comprising, inadmixture with a diagnostically acceptable carrier, an amount of mousemonoclonal antibody effective to detect human helper T cells, saidantibody reacting with essentially all normal human peripheral helper Tcells.
 7. A diagnostic composition of matter for detection of humanhelper T cells comprising, in admixture with a diagnostically acceptablecarrier, an amount of mouse monoclonal antibody effective to detecthuman helper T cells, said antibody reacting with essentially all normalhuman peripheral helper T cells but not with normal human peripheral Bcells, null cells, or macrophages.
 8. A diagnostic composition fordetection of human helper T cells comprising, in admixture with adiagnostically acceptable carrier, a diagnostically effective amount ofmouse monoclonal antibody having the identifying characteristics of thatproduced by hybridoma ATCC CRL
 8002. 9. A diagnostic composition fordetection of human helper T cells comprising, in admixture with adiagnostically acceptable carrier, an amount of mouse monoclonalantibody effective to detect helper T cells, said antibody reacting withessentially all normal human peripheral helper T cells but not withnormal human peripheral B cells.
 10. A diagnostic composition of matterfor detection of helper T cell deficiency or excess comprising, inadmixture with a diagnostically acceptable carrier, an amount ofmonoclonal antibody effective to detect helper T cell deficiency, whichantibody:(a) reacts with essentially all normal human peripheral helperT cells (being about 55% of all normal human peripheral T cells) but notwith normal human peripheral B cells, null cells or mcarophages; (b)reacts with about 80% of normal human thymocytes; (c) does not reactwith leukemic cells from humans with T cell chronic lymphoblasticleukemia, B cell chronic lymphoblastic leukemia, T cell acutelymphoblastic leukemia, or null cell acute lymphoblastic leukemia; (d)reacts with the human T cell line CEM, but not with HJD-1, Laz 191, orHM1; (e) does not react with Epstein-Barr virus-transformed human B celllines Laz 007, Laz 156, Laz 256, or SB; (f) reacts with about 55% ofRhesus monkey peripheral T cells; (g) fixes complement; and (h) definesa T cell population which is unreactive with anti-TH2 serum and is onlyminimally cytotoxic.
 11. A method for detection of helper T cell defector excess in an individual which comprises reacting a T cell compositionfrom said individual with a diagnostically effective amount ofmonoclonal antibody and measuring the percentage of the total peripheralT cell population which reacts with said antibody, which antibody:(a)reacts with essentially all normal human peripheral helper T cells(being about 55% of all normal human peripheral T cells), but not withnormal human peripheral B cells, null cells or macrophages; (b) reactswith about 80% of normal human thymocytes; (c) does not react withleukemic cells from humans with T cell chronic lymphoblastic leukemia, Bcell chronic lymphoblastic leukemia, T cell acute lymphoblasticleukemia, or null cell acute lymphoblastic leukemia; (d) reacts with thehuman T cell line CEM, but not with HJD-1, Laz 191, or HM1; (e) does notreact with Epstein-Barr virus-transformed human B cell lines Laz 007,Laz 156, Laz 256, or SB; (f) reacts with about 55% of Rhesus monkeyperipheral T cells; (g) fixes complement; and (h) defines a T cellpopulation which is unreactive with anti-TH2 serum and is only minimallycytotoxic.
 12. The method of claim 11 wherein the helper T celldeficiency is type II acquired agammaglobulinemia.